Process for producing sugar chain derivative, structure analysis method, and sugar chain derivative

ABSTRACT

A process for preparing an oligosaccharide derivative from an oligosaccharide mixture, the process being characterized in that the process comprises the steps of (a) introducing a lipophilic group into oligosaccharides of the mixture to obtain a mixture of oligosaccharide derivatives, and (b) treating the oligosaccharide derivative mixture by serotonin affinity column chromatography.

TECHNICAL FIELD

The present invention relates to a process for preparing oligosaccharidederivatives from a mixture of oligosaccharides and a method of analyzingthe structure of oligosaccharides. The invention also relates to noveloligosaccharide derivatives produced by the process of the invention forpreparing oligosaccharide derivatives.

BACKGROUND ART

It has been thought that oligosaccharides in glycoproteins have thefunctions, for example, of retaining the stereo structure of the proteinand acquiring resistance for preventing the protein from decomposingwith proteases. It has recently been revealed that oligosaccharides inglycoproteins participate in the phenomena of life such as fertilizationand differentiation, signal transmission, canceration, intracellulartransport of proteins and control of physiological activity. Theclarification of the relationship between the bonding molecules on thesurface of cells, glycoproteinaceous hormones and like oligosaccharidesand the functions thereof has matured to the concept of glycoscienceconsortium. While the functional research on oligosaccharides haspresently been conducted chiefly on sugar transferases (oligosaccharidegenes) for effecting biosynthesis of oligosaccharides, sugartransferases are also preserved by genome information and participate inthe functions of life through cooperation with other proteins. From thisviewpoint, it is necessary to conduct functional analysis ofoligosaccharides through structural glycomics procedures for capturingand analyzing the overall picture of oligosaccharides developing in thecells and tissues.

The structural glycomics in glycoscience functions to comprehensivelyanalyze the oligosaccharide recognition mechanism which plays animportant role in many phenomena of life, and this function is anindispensable element in functional glycomics. The technical factorsrequired of structural glycomics are high comprehensiveness, highthroughput, high sensitivity and high precision.

The structure of oligosaccharides in glycoprotein is presently analyzedby labeling oligosaccharides cut out from a protein with a fluorescentmaterial and thereafter analyzing the oligosaccharides by highperformance liquid chromatography (HPLC) and mass spectrometry (MS).This process has become useful means owing to a dramatic advance in massspectrometry (Nonpatent Literature 1 to 4), and anion exchange columnchromatography has been exclusively used for separating sialooligosaccharides (Nonpatent Literature 5).

[Nonpatent Literature 1] Biomed Chromatogr. 16:103-115(2002) [NonpatentLiterature 2] Anal Biochem. 206: 278-287(1992) [Nonpatent Literature 3]Biochem Soc Trans. 21:121-125(1993) [Nonpatent Literature 4] Chem Rev.102: 321-369(2002) [Nonpatent Literature 5] Biochim Biophys Acta. 705:167-173(1982)

However, the comprehensive analysis of oligosaccharides in cells andtissues involves the problem of the versatility in the modification ofthe nonreducing terminal of sialic acid, fucose or the like and thebranching of the oligosaccharide, so that it is impossible to fullyseparate the oligosaccharides which are present conjointly and to obtaina satisfactory result. Especially, the ion exchange column, which has nospecific separating function, not only fails to effect full separationbut also requires desalting treatment subsequent to the separationprocedure, and is therefore not practically useful.

Accordingly, it has been earnestly desired to provide a useful methodwhich is capable of fully analyzing the structure of oligosaccharideswhich is specific to particular cells or tissues, with considerationgiven to the nonuniformity in the information as to sucholigosaccharides.

An object of the present invention is to provide means for individuallyseparating and obtaining oligosaccharides from a mixture thereof likethose present in cells or tissues.

Another object of the invention is to provide means for analyzing thestructure of each oligosaccharide compound separated off.

Still another object of the invention is to provide noveloligosaccharide derivatives.

DISCLOSURE OF THE INVENTION

The present invention provides the following.

1. A process for preparing an oligosaccharide derivative from anoligosaccharide mixture, the process being characterized in that theprocess comprises the steps of (a) introducing a lipophilic group intooligosaccharides of the mixture to obtain a mixture of oligosaccharidederivatives, and (b) treating the oligosaccharide derivative mixture byserotonin affinity column chromatography.

2. A process for preparing an oligosaccharide derivative described abovewherein the step (b) is followed by the step (c) of conducting normalphase chromatography with use of an amino column or amide column.

3. A process for preparing an oligosaccharide derivative described abovewherein the step (c) is preceded by the step (d) of treating theresulting eluate with a glycosidase.

4. A method of analyzing the structure of an oligosaccharide in anoligosaccharide mixture, the process being characterized in that theprocess comprises the steps of (a) introducing a lipophilic group intooligosaccharides of the mixture to obtain a mixture of oligosaccharidederivatives, (b) treating the oligosaccharide derivative mixture byserotonin affinity column chromatography, and (e) treating the resultingeluate by a mass spectrometric method.

5. A method of analyzing the structure of an oligosaccharide describedabove wherein the step (b) is followed by the step (c) of conductingnormal phase chromatography with use of an amino column or amide column.

6. A method of analyzing the structure of an oligosaccharide describedabove wherein the step (c) is preceded by the step (d) of treating theresulting eluate with a glycosidase.

7. A method of analyzing the structure of an oligosaccharide accordingto par. 4 wherein the mass spectrometric method comprise MALDI-TOF MS.

8. Oligosaccharide derivatives of the formulae (1) to (6) given belowwherein R¹ is 2-caboxyphenyl, 3-carboxyphenyl, 4-carboxyphenyl,p-ethoxycarbonylphenyl or 2-pyridyl, R² is hydroxyl, the group -Asn orthe group -Asn-R³ wherein Asn is an asparagine group, R³ is acarbamate-type or amide-type protective group, and Ac is acetyl.

9. A cancer marker derived from an oligosaccharide derivative of one ofthe formulae (1) to (6).

We have conducted intensive research and treated oligosaccharides byintroducing a liophilic group into the oligosaccharides to obtainoligosaccharide derivatives, and subjecting the derivatives to affinitycolumn chromatography wherein serotonin having affinity for sialic acidserves as a ligand. Consequently, we have found that asialooligosaccharides can be separated from sialo oligosaccharides by thistreatment, and that the sialo oligosaccharides can be separated furtherinto monosialo, disialo, trisialo and tetrasialo oligosaccharidesaccording to the number of sialic acid residues.

We have further found that when the fractions obtained by the affinitycolumn chromatography are subjected to chromatography using an aminocolumn or amide column, oligosaccharides which are different in branchedstructure can be obtained as separated meticulously. This makes itpossible to produce a large quantity of oligosaccharide of singlestructure.

We have further found that when the oligosaccharide derivativesseparated off are treated by causing a suitable glycosidase to act oneach derivative, subjecting the reaction mixture to chromatography usingan amino column or amide column for isolation and subjecting theresulting oligosaccharide derivative to mass spectrometry, the structureof oligosaccharides can be analyzed comprehensively with high precision.Thus the present invention has been accomplished.

The oligosaccharides of the oligosaccharide mixture to be used in thepreparation process are not limited particularly but includeasparagine-linked oligosaccharides (N-glycoside-linkedoligosaccharides), mucin-type oligosaccharides (O-glycoside-linkedoligosaccharides), free-type oligosaccharides and furtheroligosaccharides having an amino acid attached thereto, such asoligosaccharide-linked asparagine.

These oligosaccharides may be those prepared by a chemical method. Forexample oligosaccharides derived from natural glycoproteins are in theform of a mixture of oligosaccharides which are randomly deficient inthe sugar residue at the nonreducing terminal, preferable to use is amixture of such oligosaccharides. Also preferable to use is anoligosaccharide mixture including oligosaccharides having a sialic acidresidue.

Examples of mixtures of oligosaccharides are oligosaccharide mixturesderived from natural materials such as milks, bovine-derived fetuin,eggs, or cells and tissues of living bodies. It is especially desirableto use oligosaccharides derived from cancer tissues or cancer cellssince results of great interest are then expectable.

Examples of preferred mixtures of natural oligosaccharides are thosegiven below, among which oligosaccharide mixtures including sialooligosaccharides are desirable.

It is possible to use a mixture of oligosaccharide-linked asparagineswhich is prepared by obtaining a mixture of glycoproteins and/orglycopeptides from a natural material by a known method, causing aprotease or the like to act on the mixture to cut off peptide portionsand purifying the cut-off portions by chromatography with use of a gelfiltration column or ion exchange column.

It is also possible to use a mixture of oligosaccharides which isobtained by homogenizing tissues or cells in an incubation medium usingtissues or cells of a living body or incubated tissues or incubatedcells, centrifuging the homogenized mixture to obtain a cell membranefraction, treating the fraction with 2-mercaptoethyl alcohol andthereafter causing N-glycanase to act on the resulting fraction.

It is further possible to use a mixture of free oligosaccharides whichis obtained by homogenizing incubated tissues or incubated cells,centrifuging the homogenized mixture and collecting the resultingsupernatant. These oligosaccharides include neutral oligosaccharides,i.e., high mannose-type oligosaccharides or a wide variety of sialooligosaccharides and are therefore suitable for use in preparing variousoligosaccharides.

A lipophilic group is introduced into the oligosaccharides in theoligosaccharide mixture to obtain a mixture of oligosaccharidederivatives.

The lipophilic group is a substituent capable of dissolving lipids orsoluble therein and to be formed by reacting with a ring-opened aldehydeat the reducing terminal of the oligosaccharide, or with theasparagineamino group or carboxyl group of the oligosaccharide-linkedasparagine. Examples of such substituents are those usually useful asfluorescent labels, such as 2-, 3- or 4-carboxyphenylamino group,p-ethoxycarbonylphenylamino group and 2-pyridylamino group, andsubstituents for use as carbamate-type or amide-type protective groups,such as 9-fluorenylmethoxycarbonyl (Fmoc) group, tert-butoxycarbonyl(BOC) group, benzyl, allyl, allyloxycarbonyl and acetyl.

These lipophilic groups can be introduced into oligosaccharides by aknown method. Preferable to use is 2-carboxyphenylamino group, Fmocgroup or BOC group in view of ease of handling and the stability of theoligosaccharide to be obtained, and because the excitation lightcorresponds to a mercury light source or laser light source.

For example, an aminoalditol derivative can be prepared by reacting2-aminobenzoic acid with an oligosaccharide in the presence of areducing agent such as sodium cyanoborohydride or (dimethylamino)borane.

Further for example, 9-fluorenylmethyl-N-succinimidyl carbonate can bereacted with oligosaccharide asparagine in the presence of sodiumhydrogencarbonate, whereby Fmoc group can be introduced into theasparagine, as attached to the amino group of the asparagine in themanner of carbamate.

The procedures described above afford mixtures of oligosaccharidederivatives having a lipophilic group introduced therein.

The mixture of oligosaccharide derivatives obtained is subjected toserotonin affinity column chromatography for separation.

An affinity column wherein serotonin having affinity for sialic acidserves as a ligand is used for the serotonin affinity chromatography tobe conducted in the present invention.

The serotonin affinity column may be prepared by immobilizing serotoninto a filler material, or a column commercially available may be used. Anexample of commercial column is LA-Serotonin Column (product of J-OILMILLS, INC.). The separation conditions for chromatography are suitablydetermined. For example, linear gradient elution can be conducted forseparation using a fluorescent detector at an excitation wavelength of350 nm, fluorescent wavelength of 425 nm and flow rate of 0.5 ml/min,and using a mobile phase comprising ultrapure water and aqueous solutionof ammonium acetate.

The mixture of oligosaccharide derivatives can be separated according tothe number of sialic acid residues in the oligosaccharide derivatives.First eluted are asialo oligosaccharide derivatives having no sialicacid residues, subsequently eluted are monosialo oligosaccharidederivatives and thereafter eluted are disialo derivatives. Thus eluatesare separately obtained in proportion to the increase in the number ofsialic acid residues.

The oligosaccharide derivatives thus separated by the serotonin affinitycolumn are treated by normal phase HPLC using a polymer-base aminocolumn or silica-base amide column, whereby the oligosaccharidederivatives can be separated from one another meticulously. The term“normal phase chromatography” refers to a chromatographic procedurewherein a polar solid phase of amino group, aminopropyl group oracrylamide group is used as the filling material. This procedure ischaracterized by the separation effected based on the difference in thedegree of distribution of the sample components to the solid phase andmobile phase. Basically this mode of separation is based on thehydrophilic properties of oligosaccharides. This mode of chromatographyis usable favorably also for the separation of isomers ofoligosaccharides having sialic acid attached thereto. The procedure isusable also favorably for the separation of asialo oligosaccharideswhich are treated with a dilute acid or neuraminidase.

The polymer-base amino column to be used may be a column prepared by theuser and filled with a stationary phase which comprises a polymer, suchas polyvinyl alcohol-base polymer gel, having amino group attachedthereto, whereas a commercial column is usable.

The amino column commercially available is, for example, Asahi ShodexNH2P-504E (product of Showa Denko K.K.). An example of commercial amidecolumn is TSK-GEL Amide-80 (product of TOSOH Corp.).

The separation conditions for chromatography are suitably determined.For example, linear gradient elution can be conducted for separationusing a fluorescent detector at an excitation wavelength of 350 nm,fluorescent wavelength of 425 nm and flow rate of 1 ml/min, and using amobile phase comprising acetonitrile containing acetic acid and aqueoussolution containing acetic acid and triethylamine.

The oligosaccharide structure of the oligosaccharide derivative thusobtained by isolation can be analyzed by the application of glycosidaseand mass spectrometry.

The glycosidase to be used can be a known one. Examples of such enzymesusable are sialidase, galactosidase, mannosidase, N-acetylglucosamidase,fucosidase, etc.

The mass spectrometry can be conducted by a mass spectrometer forpracticing a conventional known mass spectrometric method. Themeasurement may preferably be conducted by MALDI-TOF MS that is usedespecially for oligosaccharide analysis in recent years.

The structure of oligosaccharides is analyzed by causing a specifiedglycosidase to act on the oligosaccharide, thereafter treating thereaction mixture by normal phase HPLC using a polymer-base amino columnor a silica-base amide column, subjecting the resulting fraction to massspectrometry with consideration given to a loss of mass andcharacteristics of hydrolase, and repeating these steps.

The lipophilic group is removed from the oligosaccharide derivativeobtained. In this way, various oligosaccharides can be artificiallyobtained easily in large quantities.

The lipophilic group can be removed by a conventional known method.

For example, 2-carboxyphenylamino group can be removed by reactinghydrogen peroxide with the oligosaccharide derivative in acetic acid atroom temperature, whereby a free-type oligosaccharide can be collectedeasily.

Fmoc group is removable by reacting morpholine with the oligosaccharidederivative in N,N-dimethylformamide. BOC group is removable by reactinga weak acid with the oligosaccharide derivative.

In the case where the oligosaccharide is oligosaccharide-linkedasparagine, the asparagine residue is removable, for example, byreacting anhydrous hydrazine with the asparagine and thereafteracetylating the reaction mixture, or by refluxing the asparagine in abasic aqueous solution with heating and thereafter acetylating thereaction mixture.

Such oligosaccharides are very useful in the field of developingpharmaceuticals. For example, these oligosaccharides are useful for thesynthesis of cancer vaccines. The oligosaccharide obtained may besubjected to a combination of chemical reactions and reactions withsugar transferases and thereby made into a derivative wherein new sugarresidues are added to the oligosaccharide for the development of a novelvaccine.

The structure analyzing method and the preparation process of thepresent invention have made it possible for us to isolate theoligosaccharides of the formulae (1) to (6) given below which have notbeen found in various cancer cells.

wherein R¹ is 2-caboxyphenyl, 3-carboxyphenyl, 4-carboxyphenyl,p-ethoxycarbonylphenyl or 2-pyridyl, R² is hydroxyl, the group -Asn orthe group -Asn-R³ wherein Asn is an asparagine group, R³ is acarbamate-type or amide-type protective group, and Ac is acetyl.

It is thought that these novel oligosaccharides appear specifically incancer cells, and such oligosaccharides can be utilized as cancermarkers.

For example, a polyclonal antibody or monoclonal antibody is prepareedwhich specifically recognizes a specific oligosaccharide in cancercells, for use in detecting the oligosaccharide by an immunologicaltechnique.

For the preparation of polyclonal antibody, the oligosaccharide or ahapten thereof is combined with a protein or like high molecularcompound (carrier) to serve as an antigen, which is used toimmunologically sensitize a mammal, such as mouse, hamster, guinea pig,chicken, rat, rabbit, canine, goat, sheep or bovine, and blood iscollected from the mammal to obtain an antiserum containing a polyclonalantibody.

A hybridoma which is obtained by the fusion, for example, ofantibody-producing cells and myeloma cell strain is incubated to producea monoclonal antibody, which is then purified.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an affinity column chromatogram of oligosaccharide derivativesobtained in Example 1.

FIG. 2 are affinity column chromatograms of oligosaccharide derivativesobtained in Example 2.

FIG. 3 are chromatograms of HPLC of oligosaccharide derivatives obtainedin Example 3.

FIG. 4 are chromatograms of HPLC of oligosaccharide derivatives obtainedin Example 3.

FIG. 5 are chromatograms of HPLC of oligosaccharide derivatives obtainedin Example 3.

FIG. 6 are chromatograms of HPLC of oligosaccharide derivatives obtainedin Example 3.

FIG. 7 are chromatograms of HPLC of oligosaccharide derivatives obtainedin Example 3.

FIG. 8 is of oligosaccharide derivatives obtained in Example 4.

FIG. 9 is a chromatogram of HPLC of oligosaccharide derivatives obtainedin Example 5.

FIG. 10 is an affinity column chromatogram of oligosaccharidederivatives obtained in Example 6.

FIG. 11 is an affinity column chromatogram of oligosaccharidederivatives obtained in Example 7.

FIG. 12 are chromatograms of HPLC of oligosaccharide derivativesobtained in Example 7.

BEST MODE OF CARRYING OUT THE INVENTION

Reference Examples and Examples are given below. However, the inventionis not limited to the examples given below.

EXAMPLE 1 Separation of Human Serum-Derived [1-Acidic Glycoprotein(AGP)] Oligosaccharides by Serotonin Affinity Chromatography

One mg of human serum-derived AGP (product of Sigma-Aldrich Japan) wasdissolved in 50 μl of 20 mM phosphoric acid buffer solution (pH 7.5),N-glycanase F (2 units, 4 μl) was added to the solution, and the mixturewas reacted at 37° C. for 12 hours. The resulting reaction mixture wasboiled at 100° C. for 3 minutes and centrifuged, and the supernatant wascollected.

To the collected supernatant was added 100 μl of a solution obtained bydissolving 2-aminobenzoic acid (2-AA) and sodium cyanoborohydride eachto a concentration of 3% in a mixture (500 μl) of 2% of boric acid and4% of sodium acetate, followed by a reaction at 80° C. for 1 hour. Thereaction mixture was fractionated using a Sephadex LH-20 column (0.7 cm,i.d., 30 cm) as equilibrated with a 50% aqueous solution of methanol,the resulting fractions were measured using a spectrophotometer (productof Hitachi, Ltd., Model F-4010) at an excitation wavelength of 335 nmand fluorescent wavelength of 410 nm, and the fluorescent fractioneluted first was collected as a mixture of oligosaccharide derivatives.

The mixture obtained was subjected to serotonin affinity columnchromatography to obtain separated oligosaccharide derivatives. FIG. 1shows the result of separation by the affinity column chromatography.

Conditions for serotonin affinity column chromatographyColumn: LA-Serotonin column (4.6×150 mm, product of Japan Oil Mills)

Pump: JASCO Model PU-980

Flow rate: 0.5 ml/minDetector: JASCO Model FP-920 fluorescent detectorExcitation wavelength: 350 nmFluorescent wavelength: 425 nmMobile phase: ultrapure water used as solution A, and 40 mM ammoniumacetate aqueous solution as solution BGradient conditions: Linear gradient elution was conducted using 5% ofsolution B for 2 minutes after the sample was poured in and using theammonium acetate solution so that the concentration thereof would be 30mM 37 minutes later and subsequently 40 mM 10 minutes later.

The same conditions as above were used in the following examples forseparation by serotonin affinity chromatography.

EXAMPLE 2

Separation of Human Cancer Cell-Derived Oligosaccharides by SerotonineAffinity Chromatography

Cell Incubation

Used for incubation were human renal adenocarcinoma cells ACHN, humanlung cancer cells A549, human gastric cancer cells MKN45 and human celllymphocytic lymphoma U937. Cells were incubated in cell cultivationdishes in the presence of 5% CO₂ at 37° C., using DMEM (Dulbecco'sModified Eagle Medium, product of Sigma-Aldrich Japan) containing 10% ofbovine serum [newborn calf serum (NCS), product of Sigma-Aldrich Japan]as immobilized by being heated at 50° C. for 30 minutes in advance forACHN and A549 and using RPMI-1640 (product of Sigma-Aldrich Japan)containing 10% of NCS for U837 and MKN45. The cells other than U937 werewashed as held in an 80% confluent state with an isotonic phosphoricacid buffer (PBS) during cultivation and then treated at 37° C. for 5minutes with addition of trypsin, and the cells separated off werecollected, subsequently washed with PBS and thereafter subcultured.

Preparation of Cell Membrane Fraction

Cells in 80% confluent state for use in preparing a cell membranefraction were collected from the incubator using a cell scraper. Thecells collected were washed with PBS and homogenized with a glasshomogenizer in 10 mM Na₂HPO₄ (pH 7.5) containing 1% protease inhibitorto a concentration of 1×10⁵ cells/5 ml, 10 ml of 20 mM Tris-HCl buffer(pH 7.5) containing 0.5 M sucrose was then added to the mixture, theresulting mixture was centrifuged at 3000 rpm, 4° C. for 15 minutes, andthe supernatant was thereafter collected and centrifuged at 19000 rpm,4° C. to obtain a precipitate as a cell membrane fraction.

Separation of Oligosaccharides from the Cell Membrane Fraction

Added to the cell membrane fraction (1×10⁷ cells) were 40 μl of 1% SDSsolution first and then 2-mercaptoethyl alcohol to a concentration of1%, and the mixture was thereafter heated on a water bath boiling at100° C. for 5 minutes for solubilization. The solution containing themembrane fraction was cooled to room temperature, NP-40 was added to aconcentration of 1%, and phosphoric acid buffer (pH 7.5) was added tothe mixture to a final concentration of 20 mM. With addition of 4 μl ofN-glycanase F (2 units, product of Roche Diagnostics), the mixture wasincubated at 37° C. overnight and boiled on a water bath boiling at 100°C. for 5 minutes, 95% ethanol was added to the mixture to a finalconcentration of 75%, the resulting mixture was centrifuged at 15000rpm, 4° C., and the supernatant was treated in a vacuum to dryness toobtain oligosaccharides derived from the cell membranes.

In the same manner as in Example 1, 2-AA was introduced into theoligosaccharides, followed by serotonin affinity column chromatographyto obtain oligosaccharide fractions. FIG. 2 shows the result ofseparation by the column chromatography.

EXAMPLE 3 Separation of Cancer Cell-Derived Oligosaccharides by NormalPhase Chromatography and Structural Analysis

The cancer cell-derived oligosaccharide derivatives (corresponding to1×10⁷ cells) of each fraction obtained in Example 2 were dissolved in 20μl of 20 mM acetic acid buffer (pH 5.0), 4 μl of sialidase (2 mU,product of Marukin Bio) was added to the solution, and the mixture wasreaction at 37° C. for 24 hours. The reaction mixture was boiled at 100°C. for 3 minutes and centrifuged to obtain a supernatant.

The resulting supernatant was subjected to normal phase HPLC using anamide column to obtain oligosaccharide fractions. FIGS. 3 to 7 show theresult of separation by HPLC.

Conditions for HPLC Column: TSK-GEL Amide-80 (TOSOH CORPORATION, Japan,4.6×250 mm)

Column temperature: 40° C.

Pump: JASCO Model PU-980

Flow rate: 1 ml/minDetector: JASCO Model FP-920 fluorescent detectorExcitation wavelength: 350 nmFluorescent wavelength: 425 nmMobile phase: acetonitrile solution containing 0.2% acetic acid andserving as solution A and aqueous solution containing 0.1% of aceticacid and 0.1% of triethylamine and used as solution BGradient conditions: Linear gradient elution was conducted using 30% ofsolution B for 2 minutes after the sample was poured in so that theamount of solution B would be 65% in 60 minutes.

Glycosidase and Structural Analysis by Mass Spectrometry

The oligosaccharide derivative of peak 31 derived from U937 wasdissolved in 20 μl of 20 mM citric acid buffer (pH 3.5), 1 μl ofβ-galactosidase (25 mU, product of Seikagaku Kogyo Co., Ltd) was addedto the solution, and the mixture was reacted at 37° C. for 24 hours. Thereaction mixture was boiled at 100° C. for 3 minutes and centrifuged tocollect the supernatant. The supernatant obtained was subjected tonormal phase HPLC using an amide column, affording a fraction, which wasanalyzed by MALDI-TOF MS. As a result, an oligosaccharide (a) wasobtained which was 2718 in molecular weight.

The oligosaccharide derivative (a) obtained was dissolved in 20 μl of 20mM citric acid buffer (pH 5.0), 1 μl of β-N-acetylhexaminidase (10 mU,product of Seikagaku Kogyo Co., Ltd) was added to the solution, and themixture was reacted at 37° C. for 24 hours. The reaction mixture wasboiled at 100° C. for 3 minutes and centrifuged to collect thesupernatant. The supernatant obtained was analyzed in the same manner asabove, affording an oligosaccharide derivative (b) which was 1906 inmolecular weight.

The oligosaccharide derivative (b) was further treated withβ-galactosidase, consequently affording an oligosaccharide derivative(c) which was 1582 in molecular weight.

These results revealed that the peak 31 was the oligosaccharide of theformula given below.

Similarly, the oligosaccharide derivative of peak 35 was treated withβ-galactosidase, β-N-acetylhexaminidase and then with β-galactosidase,whereby the derivative was found to be the oligosaccharide of theformula given below.

Using suitable hydrolases, MALDI-TOF MS was conducted for thederivatives of other peaks. Tables 1 to 5 show oligosaccharidestructures corresponding to the peaks shown in FIGS. 3 to 7. Themolecular weights given in Tables 1 to 14 are those (MW) of compoundswherein 2-aminobenzoic acid is attached to the reducing terminal of theoligosaccharide. The symbols resent the following.

Gal: D-galactose, GlcNAc: N-acetylglucosamine, Man: D-mannose, Fuc:fucose, 2-AA: 2-aminobenzoic acid, NeuAc: sialic acid.

The oligosaccharide wherein 2-aminobenzoic acid is attached to thereducing terminal thereof, for example, the structure of theoligosaccharide portion represented by the formula given below will berepresented by -4GlcNAcβ1-4GlcNAc-2-AA.

MALDI-TOF MS analysis

The device used was Voyager DE-PRO (PE Biosystems, Framingham, Mass.).The measurement was conducted in linear/negative ion mode at anacceleration voltage of 20 kV and grid voltage of 96.3% with a delaytime of 1000 nsec, lens offset 1.25 and laser intensity (nitrogen laser)of 2700. A 0.5 μL quantity of the sample as dissolved in water waskneaded with 0.5 μL of 20 mg/mL methanol solution of2,5-dihydroxybenzoic acid (DHB), and the mixture was dried to obtain asample for use in measurement.

TABLE 1 Peak No. MW Structure 1 1029

2 1176

3 1192

4 1379

5 1395

6 1354

7 1541

8 1516

9 1582

10  1678

11  1785

TABLE 2 Peak No. MW Structure 12 1760

13 1840

14 1906

15 2002

16 2052

17 2125

18 2116

19 2636

20 3001

TABLE 3 Peak No. MW Structure 21 1557

22 1598

23 1743

24 2270

25 2417

26 2491

27 2563

28 2782

TABLE 4 Peak No. MW Structure 29 2928

30 3147

31 3366

32 1921

33 2109

34 3731

TABLE 5 Peak No. MW Structure 35 4096

36 4461

37 4826

38 5191

39 5556

40 2474

EXAMPLE 4 Free Oligosaccharide 1 Present in Cells

Human gastric cancer cells MKN 45 were washed with PBS and homogenizedwith a glass homogenizer in 10 mM Na₂HPO₄ (pH 7.5) containing 1%protease inhibitor to a concentration of 1×10⁸ cells/5 ml, 10 ml of 20mM Tris-HCl buffer (pH 7.5) containing 0.5 M sucrose was then added tothe mixture, the resulting mixture was centrifuged at 3000 rpm, 4° C.for 15 minutes, and the supernatant was thereafter collected andcentrifuged at 19000 rpm, 4° C. The resulting supernatant was treated ina vacuum to dryness to obtain a free-type oligosaccharide mixture.

In the same manner as in Example 1, 2-AA was introduced into theoligosaccharide mixture to obtain a mixture of free-typeoligosaccharides, which was fractionated by serotonin affinity columnchromatography to obtain free-type oligosaccharide derivatives.

FIG. 8 shows the result of separation by the affinity columnchromatography. Each fraction obtained was separated by normal phaseHPLC using an amino column to obtain a free-type oligosaccharidederivative.

Conditions for HPLC Column: Asahi Shodex NH2P-50 4E (Showa Denko, Tokyo,Japan, 4.6×250 mm)

Column temperature: 50° C.

Pump: JASCO Model PU-980

Flow rate: 1 ml/minDetector: JASCO Model FP-920 fluorescent detectorExcitation wavelength: 350 nmFluorescent wavelength: 425 nmMobile phase: acetonitrile solution containing 2% acetic acid andserving as solution A and aqueous solution containing 5% of acetic acidand 3% of triethylamine and used as solution BGradient conditions: Linear gradient elution was conducted using 30% ofsolution B for 2 minutes after the sample was poured in so that theamount of solution B would be 95% in 80 minutes. Solution B wasmaintained in an amount of 95% for 100 minutes.

The free-type oligosaccharide derivative obtained was suitably treatedwith glycosidase (sialidase, α-mannosidase, β-galactosidase,β-acetylhexaminidase, etc.), followed by normal phase HPLC forseparation using the above-mentioned amino column. The fractionsobtained were lyophilized and analyzed by MALDI-TOF MS to determine thestructure of the oligosaccharide derivative.

The treatment with α-mannosidase is conducted by dissolving theoligosaccharide derivative in 20 μl of 20 mM citric acid buffer (pH4.5), adding 2 μl of α-mannosidase (10 mM, product of Seikagaku KogyoCo., Ltd.), reacting the mixture at 37° C. for 24 hours, boiling thereaction mixture at 100° C. for 3 minutes, centrifuging the mixture andcollecting the supernatant. The treatment with the other hydrolases wereconducted in the same manner as in the foregoing example. Tables 6 and 7show the oligosaccharide derivatives obtained.

The free oligosaccharides given in Tables 6 and 7 are novel compounds.For example, oligosaccharide derivative No. 1 which is 1321 in molecularweight is represented by the formula given below.

TABLE 6 MW Structure 1321

1483

1686

2140

2343

2505

2795

2999

3160

TABLE 7 MW Structure 3451

3816

EXAMPLE 5

A mixture of free-type oligosaccharides was obtained by the sameprocedure as in Example 4 with the exception of using human T celllymphoma Jurkat 27 (oligosaccharide-introduced cell strain) in place ofhuman gastric cancer cells MKN45.

In the same manner as in Example 1, 2-AA was introduced into thefree-type oligosaccharide mixture obtained to prepare a mixture offree-type oligosaccharide derivatives, which was fractionated byserotonin column chromatography to obtain free-type oligosaccharidederivatives

Each fraction obtained was separated by normal phase HPLC using an amidecolumn to obtain a free-type oligosaccharide derivative.

Conditions for HPLC Column: TSK-GEL Amide-80 (TOSOH CORPORATION, Japan,4.6×250 mm)

Column temperature: 40° C.

Pump: JASCO Model PU-980

Flow rate: 1 ml/minDetector: JASCO Model FP-920 fluorescent detectorExcitation wavelength: 350 nmFluorescent wavelength: 425 nmMobile phase: acetonitrile solution containing 0.1% acetic acid andserving as solution A and aqueous solution containing 0.2% of aceticacid and 0.2% of triethylamine and used as solution BGradient conditions: Linear gradient elution was conducted using 30% ofsolution B for 2 minutes after the sample was poured in so that theamount of solution B would be 65% in 60 minutes.

The free-type oligosaccharide derivatives obtained were analyzed by thesame procedure as in Example 4 to determine the structure. Table 8 showsthe oligosaccharide derivatives obtained.

TABLE 8 Peak No. Structure 1

2

3

4

5

EXAMPLE 6 Oligosaccharides of Human Cervical Cancer Cells

Human cervical cancer cells HeLa were incubated using DMEM containing10% of NCS immobilized in advance by being heated at 50° C. for 30minutes. In 80% confluent state, the cells being incubated were washedwith PBS and then treated at 37° C. for 5 minutes with addition of atrypsin solution, and the cells separated were collected, washed withPBS and thereafter subcultured. The same procedures as in Example 2 wererepeated for the preparation of a cell membrane fraction and separationof oligosaccharides from the cell membrane fraction to obtain anoligosaccharide mixture derived from cell membranes. In the same manneras in Example 1, 2-AA aws introduced into the mixture, followed byserotonin affinity chromatography to obtain oligosaccharide derivativefractions.

FIG. 10 shows the result of separation by the column chromatography.

The asialo oligosaccharide derivative fraction, monosialooligosaccharide derivative fraction and disialo oligosaccharidederivative fraction were treated with sialidase and thereafter subjectedto normal phase HPLC using an amino column for separation to obtainoligosaccharide derivatives The same conditions as in Example 4 wereused for HPLC.

The oligosaccharide derivatives obtained were suitably treated withglycosidase (sialidase, α-mannosidase, β-galactosidase,β-acetylhexaminidase, etc.), followed by normal phase HPLC forseparation using the above-mentioned amino column. The fractionsobtained were lyophilized and thereafter analyzed by MALDI-TOF MS todetermine the structure of the oligosaccharide derivatives.

Tables 9 to 12 show the oligosaccharide derivatives obtained.

TABLE 9 asialo oligosaccharide derivative MW Structure 1354

1516

1678

1760

1840

1906

2002

TABLE 10 monosialo oligosaccharide derivative MW Structure 1686

1889

2051

2197

2254

2343

2400

2546

TABLE 11 disialo oligosaccharide derivative-1 MW Structure 2342

2545

2691

2748

2894

3056

3113

TABLE 12 disialo oligosaccharide derivative-2 MW Structure 3259

3405

3421

3624

EXAMPLE 7 Oligosaccharides of Cancer Cell-Specific Antigen CD98Preparation of CD98-HC by Immune Sedimentation

Protein A-Agarose (50 μl, product of Sigma-Aldrich Japan) was washedwith 200 μl of PBS. 50 μl of PBS and 10 μg/10 μl of anti-CD 98 antibodywere added to the agarose, and the mixture was reacted at roomtemperature for 60 minutes. The resation mixture was washed with 1 ml ofPBS to remove the unadsorbed component to obtain Agarose havinganti-CD98 antibody immobilized thereon. To the agarose was added amembrane fraction (2×10⁷ cells) of HeLa cells solubilized with 400 μl of1% NP-40, and the mixture was incubated overnight at 4° C. using arotary shaker. The culture was washed with 1 ml of PBS, the unadsorbedcomponent was removed, followed by centrifuging. To the agarose havinganti-CD98 antibody immobilized thereon was added 20 μl of a 9:1 mixtureof dissociation solution (250 mM Tris-HCl buffer pH 6.8/4.6% SDS, 20%glycerin) and 2-mercaptoethyl alcohol, the mixture was boiled for 5minutes and centrifuged at 15000 rpm, and the supernatant was taken asCD98-HC and subjected to PAGE.

SDS Polyacrylamide Gel Electrophoresis

The gel electrophoresis device and power source used were products ofBio Rad. The electrophoresis was conducted using 7.5% gel and a bufferof 25 mM Tris, 198 mM glycine and 1% (w/v) SDS, at 5 mA per sheet of gelfor the first 1 hour and subsequently at 10 mA to the lower side portionof the gel.

Coomassie Brilliant Blue Staining

After SDS-PAGE, proteins were stained in a solution of 40% (v/w)methanol, 10% (v/w) acetic acid/0.2% Coomassie Brilliant Blue R-250. Onehour later, the proteins were decolored with a mixture of methanol,acetic acid and water (4:1:5)

Western Blot

The protein sample in the gel resulting from SDS-PAGE was transferredonto the PVDF membrane using Semi-Dry Blotting Device (Trans-Blot SDcell, product of BIO-RAD). Before use, the PVDF membrane was immersed inmethanol for 60 seconds and thereafter immersed in 48 mM Tris, 39 mMglycine, 20% methanol (pH 9.0) for 1 hour. The transfer was conductedwith the application of voltage for 1 hour at a constant current of 100mA. After the transfer, a blocking procedure was performed for the PVDFmembrane using PBS containing 5% skim milk and 0.05% Tween 20, and 0.05%Tween 20/PBS (5 ml) containing 5 μg of anti-CD98 antibody was thereafteradded to the membrane, followed by a reaction overnight. After thereaction, the PVDF membrane was washed four times with PBS (20 ml)containing 0.05% Tween 20. Subsequently added to the membrane was 5 mlof 0.05% Tween 20/PBS mixture containing 5 μl of HRP-labeled Protein A,followed by a reaction for 1 hour. The PVDF membrane was then washedfour times with PBS (20 ml) containing 0.05% Tween 20. To the membranewere then added 20 ml of 0.0031% hydrogen peroxide solution (100 mMTris-HCl buffer 7.5 in pH) and containing 0.05% DAB(3,3-diaminobennzidrine tetrahydrochloride) for color development.

Intragel Digestion with N-glycanase

After bands were recognized by CBB staining, the discolored solution wasreplaced by water, and desired bands were cut out and placed into anEppendorf tube. With addition of 100 μl of acetonitrile, the tube wasthen allowed to stand for 30 minutes to thereby remove water from thegel. After the removal of the acetonitrile, 100 μl of Trsi-HCl buffer7.5 in pH and containing 2 units of N-glycanase F was placed in, themixture was incubated overnight at 37° C., and oligosaccharides were cutout. Subsequently, the extract was collected, 200 μl of water was addedthereto, the mixture was stirred for 30 minutes, and an oligosaccharidemixture was obtained from the gel.

In the manner as in Example 1, 2-AA was introduced into theoligosaccharide mixture thus obtained, and the resulting mixture wastreated by serotonin affinity column chromatography to collect fractionsof oligosaccharides.

FIG. 11 shows the result of separation by the column chromatography.

The monosialo oligosaccharide derivative fraction and the disialooligosaccharide derivative fraction obtained were treated with sialidaseand thereafter treated by normal phase HPLC using an amino column toobtain oligosaccharide derivatives. The same conditions as in Example 4were used for HPLC. FIG. 12 shows the result of separation by HPLC.

The oligosaccharide derivatives obtained were suitably treated withglycosidase (sialidase, α-mannosidase, β-galactosidase,β-acetylhexaminidase, etc.), followed by normal phase HPLC forseparation using the above-mentioned amino column. The fractionsobtained were lyophilized and thereafter analyzed by MALDI-TOF MS todetermine the structure of the oligosaccharide derivatives.

Tables 13 and 14 show the oligosaccharide derivatives obtained.

TABLE 13 Peak No. MW Structure I 2051

II 2197

III 2343

IV 2400

V 2562

VI 2781

VII 2927

TABLE 14 Peak No. MW Structure VIII 2342

IX 2488

X 2545

XI 2691

XII 2853

XIII 3072

XIV 3275

INDUSTRIAL APPLICABILITY

Oligosaccharide mixtures in cells and tissues can be meticulouslyseparated and the structure of oligosaccharides can be comprehensivelyanalyzed by the processes of the invention. This serves to explore theoligosaccharides and functions thereof which still remain to beclarified. The invention is therefore expected to contribute a greatdeal to the research on oligosaccharides in the future.

1.-9. (canceled)
 10. A method of analyzing the structure of an oligosaccharide derived from cancer cells in an oligosaccharide mixture derived from cancer cells, the process being characterized in that the process comprises the steps of (a) introducing a lipophilic group into oligosaccharides of the mixture derived from cancer cells to obtain a mixture of oligosaccharide derivatives, (b) treating the oligosaccharide derivative mixture by serotonin affinity column chromatography, and (c) treating the resulting eluate by a mass spectrometric method.
 11. A method of analyzing the structure of an oligosaccharide according to claim 10 wherein the step (b) is followed by the step (c) of conducting normal phase chromatography with use of an amino column normal phase chromatography with use of an amino column or amide column.
 12. A method of analyzing the structure of an oligosaccharide according to claim 10 wherein the step (c) is preceded by the step (d) of treating the resulting eluate with a glycosidase.
 13. A method of analyzing the structure of an oligosaccharide according to claim 10 wherein the mass spectrometric method comprise MALTI-TOP MS. 14.-15. (canceled) 